1. Field of the Invention
This invention relates to the field of polypeptide ligand and receptor interactions. In particular, it relates to the field of selecting and screening antagonists and agonists for polypeptide ligands.
2. Description of the Background Art
Ligand induced receptor oligomerization has been proposed as a mechanism of signal transduction for the large family of tyrosine kinase receptors that contain an extracellular ligand binding domain (for reviews see Yarden, Y., et al., Ann. Rev. Biochem 57:443-478 (1988); Ullrich, A., et al., Cell 61:203-212 [1990]). In these models binding of one hormone molecule (or subunit) (H) per receptor (R) is thought to induce formation of an H.sub.2 R.sub.2 complex. For example, crosslinking and non-dissociating electrophoretic studies suggest that epidermal growth factor (EGF) promotes dimerization of the EGF receptor followed by receptor autophosphorylation and activation of the intracellular tyrosine kinase (Shector, Y., et al., Nature 278:835-838 (1979); Schreiber, A. B., et al., J. Biol. Chem., 258:846-853 (1983); Yarden, Y., et al., Biochemistry, 26:1434-1442 (1987); Yarden, Y., et al., Biochemistry 26:1443-1451 (1987). Studies of other tyrosine kinase receptors including the insulin receptor (Kahn, C. R., et al., Proc. Natl. Acad. Sci. U.S.A. 75:4209-4213 (1978); Kubar J., et al., Biochemistry 28:1086-1093 (1989): Heffetz, D., et al., J. Biol. Chem. 261:889-894 (1986), platelet derived growth factor (PDGF) receptor (Heldin, C. H., et al., J. Biol. Chem. 264:8905-8912 (1989); Hammacher, A., et al., EMBO J. 8:2489-2495 (1989); Seifert, R. A., et al., J. Biol., Chem. 264:8771-8778 (1989)) and insulin-like growth factor (IGF-I) receptor (Ikari, N., et al., Mol. Endocrinol. 2:831-837), indicate that oligomerization of the receptor is tightly coupled to the biological effect. Other groups have recently crystallized a polypeptide hormone in complex with its extracellular binding domain (Lambert, G., et al., J. Biol. Chem. 264:12730-12736 (1989); Gunther, N., et al., J. Biol. Chem. 265:22082-22085 (1990)). However, more detailed analyses of the structural perturbations and requirements for ligand induced changes in these or other receptors have been hampered because of the complexities of these membrane associated systems and the lack of suitable quantities of highly purified natural or recombinant receptors.
When purified receptors were available the assay procedures were often structured so that the nature of the hormone-receptor complex was not recognized. In U.S. Pat. No. 5,057,417, hGH binding assays were conducted using .sup.125 I-hGH competition with cold hGH for binding to the extracellular domain of recombinant hGH receptor (hGHbp), or hGH binding protein; the resulting complex was treated with antibody to the hGHbp, plus polyethylene glycol, to precipitate the complex formed. These immunoprecipitation assays suggested that hGH formed a 1:1 complex with hGHbp. This immunoprecipitation assay correctly detected the amount of .sup.125 I-hGH bound, but it incorrectly indicated a 1:1 molar ratio.
Various solid phase assays for hGH receptor and binding protein have been used. Such assays detected the amount of hGH bound but not the molar ratio of hGH to receptor. Binding assays with solid phase or with membrane fractions containing hGH receptor were not suitable for determining the molar ratio of hGH to receptor due to an inability to detect the total amount of active receptor and/or the amount of endogenous hGH bound. Based upon earlier work, such as with EGF, the art assumed the hGH-receptor complex would be an H.sub.2 R.sub.2 tetramer.
The hGH receptor cloned from human liver (Leung, D. W. et al., Nature, 330:537 (1987)) has a single extracellular domain (.about.28 kD), a transmembrane segment, and an intracellular domain (.about.30 kD) that is not homologous to any known tyrosine kinase or other protein. Nonetheless, the extracellular portion of the hGH receptor is structurally related to the extracellular domains of the prolactin receptor (Boutin, J. M. et al., Cell 69:(1988)) and broadly to at least eight other cytokine and related receptors. hGHbp expressed in Escherichia coli has been secreted in tens of milligrams per liter (Fuh, G., et al., J. Biol. Chem., 265:3111-3115 (1990)). The highly purified hGHbp retains the same specificity and high affinity for hGH (K.sub.D .about.0.4 nM) as compared to the natural hGHbp found in serum.
hGH is a member of an homologous hormone family that includes placental lactogens, prolactins, and other genetic and species variants of growth hormone (Nicoll, C. S., et al. Endocrine Reviews, 7:169(1986). hGH is unusual among these in that it exhibits broad species specificity and binds to either the cloned somatogenic (Leung, D. W. et al. [1987] Nature 330: 537) or prolactin receptor (Boutin, J. M., et al. Cell, 53:69). The cloned gene for hGH has been expressed in a secreted form in Eschericha coli (Chang, C. N., et al., Gene 55:189 [1987]) and its DNA and amino acid sequence has been reported (Goeddel, et al. Nature, 281:544 ([1979); Gray, et al., Gene 39:247[1985]). The three-dimensional structure of hGH has not previously been available. However, the three-dimensional folding pattern for porcine growth hormone (pGH) has been reported at moderate resolution and refinement (Abdel-Meguid, S. S. et al. Proc. Natl. Acad. Sci. U.S.A., 84:6434 [1987]). hGH receptor and antibody binding sites have been identified by homolog-scanning mutagenesis (Cunningham, et al., Science 243:1330, 1989). GH with N-terminal amino acids deleted or varied are known. See Gertler, et al., Endocrinology 118:720 (1986), Ashkenazi, et al., Endocrinology 121:414 (1987), Binder, Mol. Endo.,7:1060-1068 (1990), and WO 90/05185. Antagonist variants of hGH are described by Chen, et al., Mol. Endo., 5(10):1845 (1991) and literature set forth in the bibliography thereof; and WO 91/05853. hGH variants are disclosed by Cunningham, et al., Science, 244:1081 (1989) and Science 243:1330-1336 (1989).
Since the mode of interaction of many polypeptide ligands with their receptors has remained uncertain it has been difficult to engineer amino acid sequence variants of such ligands to achieve desired properties. Essentially, the art has introduced variation at random, perhaps in some cases with guidance from homology analyses to similarly-acting ligands or animal analogues, or from analysis of fragments, e.g., trypsin digest fragments. Then the art has screened the candidates for the desired activity, e.g., agonist or antagonist activity. The screening methods have been tedious and expensive, e.g., the use of transgenic animals (WO 91/05853). Methods are needed for improving the efficiency of selection of candidates. In particular, methods are needed for focusing on candidates likely to be either antagonists or agonists. Antagonists are substances that suppress, inhibit or interfere with the biological activity of a native ligand, while agonists exhibit greater activity per se than the native ligand.
It therefore is an object of this invention to provide improved methods for the efficient selection of agonist or antagonist polypeptide ligands.
It is another object herein to provide a method for detecting ligands that form sequential 1:2 complexes with their receptors.
Another object herein is to assay candidate substances for their ability to interfere with or promote the formation of such 1:2 ligand-receptor complexes.
An additional object is to provide amino acid sequence variants of polypeptide ligands that are capable of acting as agonists or antagonists.
Other objects, features and characteristics of the present invention will become more apparent upon consideration of the following description and the appended claims.